Vaccine Adjuvant

ABSTRACT

A Dectin-2 ligand vaccine adjuvant and a method of making and using the Dectin-2 ligand vaccine adjuvant in a vaccine to immunize a patient are disclosed. Also discloses is a vaccine composition comprising a Bl-Eng2 antigen and methods of using the vaccine composition to immunize a subject against a fungal infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application62/411,281, filed Oct. 21, 2016, which is incorporated by referenceherein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under AI093553 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND

Vaccines have been hailed as one of the greatest achievements in publichealth during the past century. The global eradication of Smallpox virusin humans and Rinderpest virus in animals, and the near eradication orsuccessful prevention of other viral or bacterial infections, forexample meningitis in children due to Hemophilus influenze Type B, offercompelling examples. Yet, the development of safe and efficaciousvaccines against fungi has been a major hurdle. This difficulty stemsfrom the relative genetic complexity and intractability of fungi in thelaboratory, limited knowledge of the mechanisms that underpinanti-fungal protective immunity, and a lack of defined antigen (Ag)candidates for vaccine protection against fungal pathogens.

To date, only two vaccines against fungi have moved into clinicaltrials. An investigational candidate vaccine containing rAls3p-N(NDV-3),directed against Candida (and also S. aureus), has been tested forsafety and immunogenicity in volunteers in a Phase I trial. Anothercandidate vaccine containing rSap2p was found to be tolerated andeffective in inducing specific antibodies and B cell memory in womenwith recurrent vulvovaginitis in a European clinical trial. Highlyconserved Ags that are shared across fungal pathogens in a family ortaxon would be preferable, but the only such component that has shownpromise is β-glucan. Cassone et. al. reported that this shared cell wallcomponent served as the basis for a glyco-conjugate vaccine againstCandida and Aspergillus. This preparation has not yet moved intoclinical trials, but β-glucan particles (GPs) could serve as anexperimental platform for the delivery of candidate vaccines againstfungi.

The incidence of fungal infections and mycoses has increasedsignificantly in the past two decades, mainly due to the growing numberof individuals who have reduced immunological function(immuno-compromised patients), such as cancer patients, patients whohave undergone organ transplantation, patients with AIDS, patientsundergoing hemodialysis, critically ill patients, patients after majorsurgery, patients with catheters, patients suffering from severe traumaor burns, patients having debilitative metabolic illnesses such asdiabetes mellitus, persons whose blood is exposed to environmentalmicrobes such as individuals having indwelling intravenous tubes, andeven in some elderly individuals. Fungal infections are often alsoattributed to the frequent use of cytotoxic and/or antibacterial drugs,which alter the normal bacterial flora. Fungi include molds, yeasts andhigher fungi. All fungi are eukaryotic and have sterols but notpeptidoglycan in their cell membrane. They are chemoheterotrophs(requiring organic nutrition) and most are aerobic. Many fungi are alsosaprophytes (living off dead organic matter) in soil and water andacquire their food by absorption. Characteristically fungi also producesexual and asexual spores. There are over 100,000 species recognized,with 100 infectious members for humans.

Human fungal infections are uncommon in generally healthy persons, beingconfined to conditions such as Candidiasis (thrush) and dermatophyteskin infections such as athlete's foot. Nevertheless, yeast and otherfungi infections are one of the human ailments which still present aformidable challenge to modern medicine. In an immuno-compromised host,a variety of normally mild or nonpathogenic fungi can cause potentiallyfatal infections. Furthermore, the relative ease with which human cannow travel around the world provides the means for unusual fungalinfections to be imported from place to place. Therefore, wild andresistant strains of fungi are considered to be one of the mostthreatening and frequent causes of death mainly in hospitalized personsand immuno-compromised patients.

The identity of conserved antigens among pathogenic fungi is poorlyunderstood.

This is especially true for immunologically significant antigens thatmay serve as immunogens to vaccinate against infection. There arecurrently no commercial vaccines against fungi despite the growingproblem of fungal infections. A vaccine against pathogenic fungi,especially one that protects against multiple fungal pathogens, would beof enormous clinical benefit, and of commercial interest. Improvedvaccines and methods of vaccination against fungi are needed in the art.

Needed in the art is an improved adjuvant for a fungal, bacterial andviral vaccines as well as novel vaccine antigens.

SUMMARY OF THE INVENTION

The present invention relates to a vaccine composition comprising aDectin-2 ligand.

In a first aspect, described herein is a vaccine suitable to immunize apatient comprising anadjuvant, wherein the adjuvant is a Dectin-2ligand. In some embodiments, the Dectin-2 ligand is a glycoprotein. Insome embodiments, the Dectin-2 ligand is Bl-Eng2. In one embodiment,Bl-Eng2 comprises SEQ ID NO:1. In some embodiments, Bl-Eng2 comprises0-linked glycosylations.

In some embodiments, the vaccine immunizes a patient against a fungalinfection. In some embodiments, the vaccine comprises glucan particles.In some embodiments, the vaccine immunizes a patient against a bacterialinfection. In some embodiments, the vaccine immunizes a patient againsta viral infection.

In a second aspect, described herein is a method of preparing a vaccinecomprising the steps of, (a) preparing a pharmaceutically acceptablevaccine stabilizer; and (b) introducing to the vaccine stabilizer asuitable antigen and an adjuvant, wherein the adjuvant is a Dectin-2ligand.

In a third aspect, described herein is a method of protecting a patientfrom an infection comprising the steps of: (a) obtaining a vaccinesuitable to immunize a patient, wherein the vaccine comprises anadjuvant and a suitable antigen, wherein the adjuvant is a Dectin-2ligand; and (b) providing a therapeutically effective amount of thevaccine to a subject, wherein the subject is protected from theinfection. In some embodiments, the infection is a fungal infection andthe patient is protected from a fungi infection. In some embodiments,the antigen is a fragment of calnexin and the fungi is selected form thegroup consisting of Histoplasma, Coccidiodes, Paracoccidioides,Penicillium, Blastomyces, Sporothrix, Aspergillus, Pneumocystis,Magnaportha, Exophiala, Neuroaspora, Cryptococcus, Schizophyllum, andCandida.

In a forth aspect, described herein is a vaccine composition comprisingBl-Eng2 and a pharmaceutically acceptable carrier. In some embodiment,Bl-Eng2 comprises SEQ ID NO:1. In some embodiments, Bl-Eng2 comprisesO-linked glycosylations. In some embodiments, the vaccine is suitable toimmunize a subject against a fungal infection. In some embodiments, thevaccine additionally comprises an adjuvant. In one embodiment, thevaccine comprises incomplete Freunds adjuvant. In some embodiment, thevaccine comprises a fragment of Bl-Eng2.

In a fifth aspect, described herein is a method of protecting a patientfrom an infection comprising the steps of: (a) obtaining a vaccinecomposition comprising Bl-Eng2 and a pharmaceutically acceptablecarrier; and (b) providing a therapeutically effective amount of thevaccine to a subject, wherein the subject is protected from theinfection. In some embodiments, the infection is a fungal infection. Insome embodiments, Bl-Eng2 comprises SEQ ID NO:1. In some embodiments,Bl-Eng2 comprises O-linked glycosylations.

BRIEF DESCRIPTION OF DRAWINGS

The patent or patent application file contains at least one drawing incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1F demonstrate identification of ligand activity and enrichmentby ConA. (A) Silver-stained SDS-PAGE gel of CWE after water wash andsonication. (B) Dectin-2 reporter cells were stimulated withplate-coated CWE treated with or without proteinase K (pro-K),α-Mannosidase (α-M), or β-Mannosidase (β-M). After 18 h, lacZ activitywas measured. Data are the mean±SD of duplicate wells. (C) Flow chart ofligand enrichment and purification. (D) CWE was incubated with ConAresin. Flow-through (FL) and eluate (E) were run on SDS-PAGE gel, silverstained and analyzed for ligand activity. (F) ConA eluate was furtherseparated by size exclusion using a BioLogic LP system (Biorad) andUltro Gel ACA44 resin (Pall Corporation) at a flow rate of 1 ml/min(blue line represents the trace line of Amo absorption). Fractions weretested by Dectin-2 reporter cells for ligand activity. Fractions 4-6contained most of the ligand activity and were separated by a second runover the size exclusion column (see FIG. 6C).

FIGS. 2A-2D show mass spec analysis identified Bl-Eng2 as a Dectin-2ligand candidate: (A) The ligand-negative and -positive fractions (#9-13and #1-7 from FIG. 6C, respectively) from the second gel filtration wereanalyzed by Mass spectrometry. Numbers on the right represent number ofpeptide specific fragments detected. (B) Domains of native B.dermatitidis Eng2 (Bl-Eng2) and recombinant Bl-Eng2 expressed in Pichiapastoris: SP denotes Signal peptide; GH16 denotes glycosyl hydrolasecatalytic domain; Ser/Thr-rich domain harbors 68 potential O-linkedglycosylatioin sites; and Myc and His tags are placed at the C terminusfor purification. (C) 0.6 μg Bl-Eng2 and 0.3 μg PDIA1 were run onSDS-PAGE gel under reducing conditions and stained for protein (left) orcarbohydrate (right). (D) Monosaccharide composition of Bl-Eng2 measuredby gas chromatography (GC). GC chromatogram of the alditolacetate-derivatized monosugars of hydrolyzed Bl-Eng2 (top).Monosaccharides are labeled as follows: Rha—rhamnose, Rib—ribose,Xyl—xylose, Man—mannose, and Glu—glucose. Unlabeled peak at 5.953 minresulted from component degradation during alditol acetatederivatization. Pie diagram shows the relative contribution ofmonosaccharides (bottom).

FIGS. 3A-3D demonstrate that Bl-Eng2 is a bona-fide, superior Dectin-2ligand. (A) Pichia-expressed proteins were plate-bound and tested forligand activity using CLR expressing B3Z reporter cells expressing FcRγchain, Dectin-2+FcRγ, MCL+FcRγ, and Mincle+FcRγ, and BWZ cells and asubline expressing Dectin-1-CD3ζ (Dectin-1). (B) Supernatants frommurine BMDCs (2×10⁵ per well) co-cultured with plate-bound Bl-Eng2 orPDIA1 were analyzed for IL-6 by ELISA. Blastomyces vaccine yeast (4×10⁵per well) was used as a positive control. (C) Supernatants from BMDCs(10⁵ per well) co-cultured with 1, 10, or 100 ng and 0.01, 0.1 or 1 pmolplate-bound Bl-Eng2, Man-LAM, Furfurman or MP98 were analyzed for IL-6by ELISA. Blastomyces vaccine yeast (10⁴, 10⁵ or 10⁶ per well) was usedas positive control. Data in A-C represent the mean±SEM of onerepresentative experiment of 3 independent experiments. (D) Bl-Eng2induces IL-6 and IL-1β by human PBMCs. Human PBMCs were stimulated withplate-bound Bl-Eng2 for 24 h and cytokines in cell culture supernatantswere measured by ELISA. Data represent the mean±SEM of 5 healthyindividuals. *, p<0.05 vs. no Bl-Eng2.

FIGS. 4A-4F show Bl-Eng2 augments CD4⁺ T cell development in vivo. Micereceived 10⁶ adoptively transferred naïve 1807 T cells prior tovaccination (A-D) or no transfer (E+F). Mice were subcutaneouslyvaccinated with 5 μg calnexin and 10 μg Bl-Eng2 or alum twice, two weeksapart, and then challenged intratracheally with B. dermatitidis 26199yeast two weeks post-vaccination. At day 4 post-infection, thefrequencies of IL-17 and IFN-γ producing 1807 T cells (A) and thenumbers of activated (CD44⁺) and cytokine-producing 1807 cells in thelung were enumerated by FACS (B). Almost all of the 1807 T cellsrecruited to the lung were CD44⁺. Data represent the average±SEM of twoindependent experiments with 8-10 mice/group. *, p<0.05 vs. control micevaccinated with calnexin and IFA alone and **, p<0.05 vs. control micevaccinated with soluble calnexin alone. Cytokines from lymph node cellsstimulated ex vivo with calnexin were measured by ELISA (D). The numberindicates the n-fold change of mice vaccinated with calnexin+Bl-Eng2 vs.mice vaccinated with calnexin alone. *, p <vs. all other groups. LungCFU were counted at day 18 post-infection when naïve mice were moribund,(C+E). *, p<0.05 vs. all other groups. Numbers reflect the n-fold changein lung CFU of mice vaccinated with calnexin and Bl-Eng2 vs. controlmice vaccinated with calnexin or IFA alone. The survival of vaccinatedmice was recorded for 30 days post-infection (FIG. 4E). *, p<0.05 vs.all other groups. At day 4 post-infection, the number ofcalnexin-specific CD4⁺ T cells was enumerated by tetramer staining (FIG.4F). Data represent the average±SEM of tetramer positive cells from oneof two independent experiments with 4-5 mice/group. *, p<0.05 vs. allother groups. Cnx denotes calnexin.

FIGS. 5A-5E show myeloid effector mechanisms by Bl-Eng-2. Mice received1807 cells prior to vaccination and were vaccinated and boosted withindicated adjuvants and formulated calnexin. Two weeks after the boost,mice were challenged i.t. with 10⁵ DsRed yeast and lungs were harvested3 days later. The percentage of dead (DsRed⁻Uvitex⁺)(blue) yeast amongtotal neutrophil-associated yeast (all Uvitex⁺ events)(blue and redtogether) (see gating strategy in FIG. 11A) were analyzed and calculated(dot plots are concatenates from 5 mice/group) to depict the amount ofkilling by neutrophils (A+B). The percentage of killing by alveolarmacrophages is shown in (C). The number of live yeast was depicted byshowing the total number of DsRed⁺ events (D) or plating lung CFU (E).The numbers indicate the n-fold reduction in live yeast (DsRed⁺ or CFU)vs. the calnexin control groups. *p<0.05 control groups withoutBl-Eng-2. Cnx denotes calnexin.

FIGS. 6A-6C show separation, characterization, and enrichment ofDectin-2 ligand activity. (A) 100 μg CWE was fractionated by a GELFREE(GF) 8100 system. The fractions were separated by SDS-PAGE and silverstained. (B) Acetone-precipitated fractions were assayed for ligandactivity. (C) Fractions 4-6 from the 1^(st) gel filtration containedmost of the ligand activity (see FIG. 1F); they were separated by asecond run over the size exclusion column (blue line represents thetrace line of Amo absorption). Fractions were tested by Dectin-2reporter cells for ligand activity. Fractions 9-13 contained most of theligand activity and were determined the positive pool; fractions 1-7were the negative pool for the subsequent mass spec analysis.

FIGS. 7A-7B show mass spec analysis identifies Bl-Eng2 as a candidateligand for Dectin-2. (A) Complete list of Mass spec candidates forDectin-2 ligands. (B) Amino acid sequence of recombinant Bl-Eng2contains 637 amino acids (SEQ ID NO:2). Colored aa match the proteindomains illustrated in FIG. 2B.

FIGS. 8A-8D demonstrate that Aspergillus Eng2 is a Dectin-2 ligand. (A)0.6 ug Pichia-expressed Aspergillus Eng-2 was plate-coated and testedfor ligand activity using CLR expressing B3Z and BWZ reporter cells. (B)30 ng plate-coated Pichia-expressed Blastomyces Eng2 and AspergillusEng2 was tested for ligand activity with Dectin-2 expressing B3Zreporter cells. (C) 30 ng plate-coated Pichia-expressed CryptococcusEng2 was tested for ligand activity with Dectin-2 expressing B3Zreporter cells. (D) Supernatants from BMDCs (2×10⁵ per well) co-culturedwith plate-coated MP98 were analyzed for IL-6 by ELISA.

FIGS. 9A-9E demonstrate that Bl-Eng2 induces the development of Th17 andTh1 cells in a Dectin-2 dependent manner and reduces lung CFUconcentration dependently. (A+C) Mice were subcutaneously vaccinatedtwice with calnexin and Bl-Eng2, two weeks apart and challengedintratracheally with B. dermatitidis 26199 yeast two weekspost-vaccination. At day 4 post-infection, the numbers of activated(CD44⁺) and cytokine producing 1807 T cells in wild type (A) andDectin-2^(−/−) mice (C) were enumerated by FACS. Data represent theaverage±SEM of 5 mice/group. *, p<0.05 vs calnexin-vaccinated controlmice. Lymph node (LN) cells from the draining brachial LN werestimulated ex vivo with calnexin and cytokines in the cell culturesupernatants were measured by ELISA (D). (B+E) At day 4 post-infection,lung CFU of (B) wild type mice and (E) Dectin-2^(−/−) mice weredetermined by plating lung homogenates. *, p<0.05 vs calnexin-vaccinatedcontrol mice. (A-E) Numbers reflect the n-fold change of mice vaccinatedwith calnexin and Bl-Eng2 vs. control mice vaccinated with calnexin. NS;not statistically significant.

FIGS. 10A-10C show that Bl-Eng2 augments adjuvancy of Alum. (A-C) Micewere subcutaneously vaccinated with 5 μg calnexin and 10 μg Bl-Eng2or/and alum twice, two weeks apart, and then challenged intratracheallywith B. dermatitidis 26199 yeast two weeks post-vaccination. At day 4post-infection, the numbers of activated (CD44⁺) and cytokine-producing1807 cells in the lung were enumerated by FACS (A+B). Data represent theaverage±SEM of 5 mice/group. *, p<0.05 vs. control mice vaccinated withcalnexin and Alum. The numbers indicate the n-fold change of micevaccinated with Alum+calnexin+Bl-Eng2 vs. mice vaccinated withAlum+calnexin. *, p<vs. all other groups. Lung CFU were counted at day 4post-infection (C). The numbers indicate the n-fold change in lung CFUof mice vaccinated with Alum+calnexin+Bl-Eng2 vs. mice vaccinated withAlum+calnexin. *, p<0.05 vs. all other groups. Cnx denotes calnexin.

FIGS. 11A-11D demonstrate gating strategy for tracking neutrophil- andalveolar macrophage-associated with yeast, activation of PMN and myeloideffector killing in the absence of 1807 T cell transfer. Viable cells(negative for fixable live/dead dye) that were Siglec F⁻, CD11b⁺, Ly6G⁺and Ly6C^(int) gated as neutrophils (PMNs) and SiglecF⁺, CD11c⁺ gated asalveolar macrophages (A). Blastomyces yeast have higher side scatterthan most leukocytes, so Uvitex⁺, SSC^(hi) neutrophils are associatedwith yeast. Phagocytes in the lungs that have phagocytosed inhaledchitin (from bedding/food) stain with Uvitex when cells arepermeabilized. The cells that have phagocytosed chitin/cellulose havedecreased Uvitex fluorescence but tend to be autofluorescent in manychannels including DsRed; an additional gate was placed on Uvitex⁺events to remove any false positives in the neutrophil gate. Activated(CD11b^(hi)) neutrophils from the neutrophil gate were calculated andshown in panel (B). Myeloid effector killing in the absence of 1807 Tcells (C+D). Mice did not receive adoptive transfer of 1807 cells priorto vaccination and were vaccinated twice with calnexin+/−Bl-Eng-2emulsified in IFA. Two weeks after the boost, mice were challenged i.t.with 10⁵ DsRed yeast and lungs were harvested 3 days later. Thepercentage of dead (DsRed⁻Uvitex⁺)(blue) among total neutrophil- ormacrophage-associated yeast (all Uvitex⁺ events)(blue and red together)(see gating strategy in FIG. 11A) were analyzed and calculated (dotplots are concatenates from 5 mice/group) to depict the amount ofkilling by PMN and macrophages (C). The number of live yeast wasdepicted by showing the total number of DsRed⁺ events or plating lungCFU (D). The number indicates the n-fold reduction in lung CFU vs. thecalnexin control group. *p<0.05 control groups without Bl-Eng-2.

FIG. 12 demonstrates a TB vaccine model. Mice were subcutaneouslyvaccinated with 5 μg Ag85B in IFA in the presence or absence of 10 μgBl-Eng-2 twice, two weeks apart. Two weeks after the boost, the micewere challenged with 150 CFU of M. tuberculosis and three weeks laterthe lungs were harvested and analyzed for T cell immune responses. Themean±SEM number of activated (CD44⁺) and cytokine producing CD4⁺ T cellswere enumerated by FACS. *, p value <0.05 vs. all other groups.

FIG. 13 demonstrates an influenza vaccine model. Mice were intranasallyvaccinated with 5 μg NP and 10 μg Bl-Eng-2 or not. 8 days after theboost, lung T cells were stimulated with NP peptide and analyzed for themean±SEM number of tetramer (NP396⁺) and cytokine producing CD8⁺ T cellsby FACS. *, p value <0.05 vs. all other groups.

FIG. 14 demonstrates that vaccination with Bl-Eng2 antigen protects miceagainst fungal infection. Mice were vaccinated subcutaneously with 5 μgBl-Eng2 protein formulated with incomplete Freunds adjuvant (IFA, whichconsists of mineral oil) twice two weeks apart. Two weeks after theboost, mice were challenged with 2×10E4 wild type (ATCC 26199) B.dermatitidis yeast. At day 4 and 11 post-infection animals weresacrificed their lungs plated for colony forming units (CFU). Datarepresent an average of 5-10 mice per group. Numbers indicate the n-foldchange vs. IFA control vaccinated mice.

FIG. 15 demonstrates T cell epitope identification of the Bl-Eng2protein. We synthesized 5 software predicted T cell epitopes (peptides)and stimulated splenocytes from vaccinated mice (FIG. 14) ex vivo.Peptide #1 (SEQ ID NO:4) stimulated splenocytes from Bl-Eng2 vaccinatedmice to produce IFN-γ comparable to full length Bl-Eng2 protein, whereasthe other peptides and Calnexin protein did not. Thus, peptide #1 (SEQID NO:4) is likely harboring the protective T cell epitope.

FIG. 16 shows a sequence alignment of Eng2 (SEQ ID NO:3) and Eng 3 (SEQID NO:12) from Aspergillus fumigatus, Eng2 (SEQ ID NO:13) fromPseudogymnoascus destructans, Eng2 (SEQ ID NO:14) from Coccidioidesimmitis, Eng2 (SEQ ID NO:15) from Coccidioides posadasii, Eng2 (SEQ IDNO:1) from Blastomyces dermatitidis, and Eng2 (SEQ ID NO:16) andHistoplasma capsulatum. The sequence of conserved peptide #1 is shaded.

DETAILED DESCRIPTION OF THE INVENTION In General

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, and patent application wasspecifically and individually indicated to be incorporated by reference.

In one embodiment, the present disclosure describes an adjuvant for usein a vaccine. The adjuvant is a Dectin-2 ligand, which stimulates animmune response when administered in a vaccine composition. In anotherembodiment, the present disclosure describes an antigen for use in avaccine. The antigen is Bl-Eng2 or a variant thereof, which stimulatesan immune response when administered in a vaccine.

Due to changes in naming conventions and related homologs, Bl-Eng2 ofthe current invention was named “Bl-Eng3” in corresponding U.S.Provisional Application No. 62/411,281, which is incorporated herein byreference. The polypeptide sequence of the novel adjuvant and antigenhas not changed.

In one embodiment, the Dectin-2 ligand is glycosylated. In oneembodiment, the Dectin-2 ligand comprises at least one N-linked glycan,O-linked glycan or combinations thereof. In one embodiment, the Dectin-2ligand comprises at least one 0-ling glycan. In one embodiment, theDectin-2 ligand is Bl-Eng2, MP98, Furfurman, or Man-LAM.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive. It is specifically contemplated that any listingof items using the term “or” means that any of those listed items mayalso be specifically excluded from the related embodiment.

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

As used herein the specification, “a” or “an” may mean one or more,unless clearly indicated otherwise. As used herein in the claims, whenused in conjunction with the word “comprising,” the words “a” or “an”may mean one or more than one.

The terms “comprise,” “have,” and “include” are open-ended linkingverbs. Any forms or tenses of one or more of these verbs, such as“comprises,” “comprising,” “has,” “having,” “includes,” and “including,”are also open-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and also covers other unlisted steps.

The terms “polypeptide,” “peptide,” and “protein,” as used herein, referto a polymer comprising amino acid residues predominantly bound togetherby covalent amide bonds. By the term “protein,” we mean to encompass allthe above definitions. The terms apply to amino acid polymers in whichone or more amino acid residue may be an artificial chemical mimetic ofa naturally occurring amino acid, as well as to naturally occurringamino acid polymers and non-naturally occurring amino acid polymers. Asused herein, the terms may encompass amino acid chains of any length,including full length proteins, wherein the amino acids are linked bycovalent peptide bonds. The protein or peptide may be isolated from anative organism, produced by recombinant techniques, or produced bysynthetic production techniques known to one skilled in the art.

The term “post-translational modification,” or “PMT” as used herein,refers to the covalent and generally enzymatic modification of proteinsduring or following protein biosynthesis. Post-translationalmodifications may occur at the C- or N-termini of the protein or on theamino acid side chains of the protein. PTMs may include, but are notlimited to, the addition of phosphates, carbohydrates, acetates, amidegroups, methyl groups, lipid molecules, and combinations thereof. Theaddition of PTM to a protein may be, but are not limited to, enzymaticphosphorylation, glycosylation, acylation, alkylation, methylation andcombinations thereof. In one embodiment of the invention, PTMs includeN- and O-linked glycosylations.

The term “glycoprotein,” as used herein, refers to a protein in which acarbohydrate, monosaccharide or glycan is attached to a hydroxyl orother functional group of the protein. The glycoprotein is the result ofa post-translational modification wherein a carbohydrate has beencovalently linked to the protein. The glycosylation may be, but is notlimited to, the covalent addition of any glucan known in the art and maybe one or more of a monosaccharide, a carbohydrate, a glucose, amannose, an N-acetylglucosamine, and combinations thereof. In oneembodiment of the invention, the glycol protein comprises at least oneO-linked glycan.

The term “therapeutically effective amount,” as used herein, refers toan amount of an antigen or vaccine that would induce an immune responsein a subject receiving the antigen or vaccine which is adequate toprevent signs or symptoms of disease, including adverse health effectsor complications thereof, caused by infection with a pathogen, such as avirus or a bacterium. Humoral immunity or cell mediated immunity or bothhumoral and cell mediated immunity may be induced. The immunogenicresponse of an animal to a vaccine may be evaluated, e.g., indirectlythrough measurement of antibody titers, lymphocyte proliferation assays,or directly through monitoring signs and symptoms after challenge withwild-type strain. The protective immunity conferred by a vaccine may beevaluated by measuring, e.g., reduction in clinical signs such asmortality, morbidity, temperature number, overall physical condition,and overall health and performance of the subject. The amount of avaccine that is therapeutically effective may vary depending on theparticular virus used, or the condition of the subject, and may bedetermined by a physician.

The term “protected,” as used herein, refers to immunization of apatient against a disease. The immunization may be caused byadministering a vaccine comprising an antigen. Specifically, in thepresent invention, the immunized patient is protected from a fungal,bacterial, or viral infection.

The term “vaccine,” as used herein, refers to a composition thatincludes an antigen. Vaccine may also include a biological preparationthat improves immunity to a particular disease. A vaccine may typicallycontain an agent, referred to as an antigen, that resembles adisease-causing microorganism, and the agent may often be made fromweakened or killed forms of the microbe, its toxins or one of itssurface proteins. The antigen may stimulate the body's immune system torecognize the agent as foreign, destroy it, and “remember” it, so thatthe immune system can more easily recognize and destroy any of thesemicroorganisms that it later encounters.

Vaccines may be prophylactic, e.g., to prevent or ameliorate the effectsof a future infection by any natural or “wild” pathogen, or therapeutic,e.g., to treat the disease. Administration of the vaccine to a subjectresults in an immune response, generally against one or more specificdiseases. The amount of a vaccine that is therapeutically effective mayvary depending on the particular virus used, or the condition of thepatient, and may be determined by a physician. The vaccine may beintroduced directly into the subject by the subcutaneous, oral,oronasal, or intranasal routes of administration.

A vaccine of the present invention will include a suitable antigen tostimulate an immune response in a subject or patient. It is envisionedthat vaccines of the present invention are not limited to a specificantigen or disease target, except where specifically specified. In someembodiments, the vaccine of the present invention provides immunityagainst a fungus, a parasite, a bacteria, a microbe, or a virus. In oneembodiment, the antigen is Bl-Eng2 or a peptide fragment thereof and thevaccine composition provides immunity against a fungus.

In some embodiments, the vaccine of the present invention providesimmunity against fungi. In one embodiment of the invention, the vaccinecomprises an antigen for the family of ascomycetes in which thepan-fungal antigen Calnexin is highly conserved, and has been shown toconfer protection against infection in experimental animal models. Anon-limiting example of an antigen of the present invention is thecalnexin fragment described in U.S. patent application Ser. No.14/203,898 (“Method of Treating Fungal Infection”) and U.S. patentapplication Ser. No. 14/643,693 (“Peptide MHCII Tetramers to DetectEndogenous Calnexin Specific CD4 T Cells”), both of which areincorporated herein in their entirety.

In some embodiments, the vaccine of the present invention providesimmunity against a Blastomyces dermatitidis infection. In oneembodiment, the vaccine comprises Bl-Eng2 as an antigen to conferprotection against a fungal infection. In some embodiments, the fungalinfection is selected from the group consisting of Blastomycesdermatitidis, Histoplasma capsulatum, Coccidioides posadasii,Coccidioides immitis, Aspergillus fumigatus and Pseudogymnoascusdestructans. In some embodiments, the Bl-Eng2 is a fragment of Bl-Eng2comprising SEQ ID NO:4. Without wishing to be bound by any particulartheory, SEQ ID NO:4 is conserved among Blastomyces dermatitidis,Histoplasma capsulatum, Coccidioides posadasii, Coccidioides immitis,Aspergillus fumigatus and Pseudogymnoascus destructans, and thisconserved sequence may be responsible for Bl-Eng2 mediated protectionagainst fungal infection.

Suitable Targets of the Present Invention

The term “fungi” or “funguses”, as used herein, refers to a member of alarge group of eukaryotic organisms that may include microorganisms,e.g., yeasts and molds. These organisms may be classified as a kingdomof fungi, which is separate from plants, animals, and bacteria. Onemajor difference between fungi and the others is that fungal cells havecell walls that contain chitin, unlike the cell walls of plants, whichcontain cellulose.

These and other differences show that the fungi form a single group ofrelated organisms, named the Eumycota (true fungi or Eumycetes), thatshare a common ancestor (a monophyletic group). This fungal group may bedistinct from the structurally similar myxomycetes (slime molds) andoomycetes (water molds). Genetic studies have shown that fungi are moreclosely related to animals than to plants. In the present invention, theterms “fungi”, “funguses”, or “fungal” may refer to fungi which maycause infection in humans and animals.

In one preferred embodiment of the present invention, fungi may includeCandida albicans (using Candida Adh1 or Als3 protein as an antigen),Aspergillus fumigatus, endemic systemic dimorphic fungi includingCoccidioides immitis and C. posadasii, Histoplasma capsulatum,Blastomyces dermatitidis, Paracoccidioides brasiliensis, Sporothrixschenkii and Penicillium marneffii) and other ascomycetes using theshared and conserved antigenic domain of Calnexin.

Aside from fungi, the present invention may be used as an adjuvant forvaccination against any infectious disease that requires the developmentof cellular immunity, in particular T helper 1 and T helper 17 CD4+cells and T cytotoxic 1 and T cytotoxic 17 CD8+ T cells. This group ofmicroorganisms may include parasites, bacteria, and viruses.

In some embodiments, the present invention may be used as an adjuvantfor vaccination against a bacterial infection. The bacteria may include,but is not limited to, Mycobacterium tuberculosis, and otherintracellular bacteria that require T cell immunity for host protection.Any suitable antigen known in the art to protect against the targetbacterial infection can be used in a vaccine composition with theadjuvant of the present invention. In one embodiment, the vaccinecomposition comprises Bl-Eng2 and Ag85B and protects against aMycobacterium tuberculosis infection.

In some embodiments, the present invention may be used as an adjuvantfor vaccination against a viral infection. The virus may include, but isnot limited to, influenza A, and other viral infections that requirecell mediated immunity for host protection. Any suitable antigen knownin the art to protect against the target viral infection can be used ina vaccine composition with the adjuvant of the present invention. In oneembodiment, the vaccine composition comprises Bl-Eng2 and nucleoprotein(NP) and protects against an influenza A infection.

Vaccine Administration

The term “administration,” as used herein, refers to the introduction ofa substance, such as a vaccine, into a subject's body through or by wayof a route that does not include the digestive tract. Theadministration, e.g., parenteral administration, may includesubcutaneous administration, intramuscular administration,transcutaneous administration, intradermal administration,intraperitoneal administration, intraocular administration, intranasaladministration and intravenous administration.

The vaccine or the composition according to the invention may beadministered to an individual according to methods known in the art.Such methods comprise application e.g. parenterally, such as through allroutes of injection into or through the skin: e.g. intramuscular,intravenous, intraperitoneal, intradermal, mucosal, submucosal, orsubcutaneous. Also, the vaccine may be applied by topical application asa drop, spray, gel or ointment to the mucosal epithelium of the eye,nose, mouth, anus, or vagina, or onto the epidermis of the outer skin atany part of the body.

Other possible routes of application are by spray, aerosol, or powderapplication through inhalation via the respiratory tract. In this lastcase the particle size that is used will determine how deep theparticles will penetrate into the respiratory tract.

Alternatively, application may be via the alimentary route, by combiningwith the food, feed or drinking water e.g. as a powder, a liquid, ortablet, or by administration directly into the mouth as a: liquid, agel, a tablet, or a capsule, or to the anus as a suppository. The term“animal-based protein”, as used herein, refers to proteins that aresourced from ruminant milk, and other sources, for example the musclemeat, of an animal, particularly a mammal. Suitable animal-basedproteins may include, but are not limited to, digested protein extractssuch as N-Z-Amine®, N-Z-Amine AS® and N-Z-Amine YT® (Sheffield ProductsCo., Norwich, N.Y.), which are casein enzymatic hydrolysates of bovinemilk.

The term “vegetable-based protein,” as used herein, refers to proteinsfrom vegetables. A vegetable-based protein may include, withoutlimitation, soy protein, wheat protein, corn gluten, rice protein andhemp protein, among others. Preferred vegetable based proteins in thepresent invention are soy proteins and corn gluten. Corn gluten is amixture of various corn-derived proteins. The soy proteins can include100% soy protein (available as VegeFuel® by Twinlab), textured soyprotein, and soybean enzymatic digest. Textured soy protein is a soyprotein that is made from defatted soy flour that is compressed andprocessed into granules or chunks. Soybean enzymatic digest describessoybean peptones that result from the partial hydrolysis of soybeanproteins.

Antibodies of the Present Invention

The term “antibody,” as used herein, refers to a class of proteins thatare generally known as immunoglobulins. The term “antibody” herein isused in the broadest sense and specifically includes full-lengthmonoclonal antibodies, polyclonal antibodies, multi specific antibodies(e.g., bispecific antibodies), and antibody fragments, so long as theyexhibit the desired biological activity. Various techniques relevant tothe production of antibodies are provided in, e.g., Harlow, et al.,ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., (1988).

The term “fusion protein,” as used herein, refers to a hybridpolypeptide which comprises protein domains from at least two differentproteins. Fusion proteins or chimeric proteins (literally, made of partsfrom different sources) are proteins created through the joining of twoor more genes that originally coded for separate proteins. Translationof this fusion gene results in a single or multiple polypeptides withfunctional properties derived from each of the original proteins.Recombinant fusion proteins are created artificially by recombinant DNAtechnology for use in biological research or therapeutics. Chimeric orchimera usually designate hybrid proteins made of polypeptides havingdifferent functions or physico-chemical patterns. Chimeric mutantproteins occur naturally when a complex mutation, such as a chromosomaltranslocation, tandem duplication, or retrotransposition creates a novelcoding sequence containing parts of the coding sequences from twodifferent genes. Naturally occurring fusion proteins are commonly foundin cancer cells, where they may function as oncoproteins. In oneembodiment of the present invention, fusion proteins comprise at leastone engineered intein.

The term “immune status” or “immunocompetence,” as used herein, refersto the ability of the body to produce a normal immune response followingexposure to an antigen. Immunocompetence is the opposite ofimmunodeficiency or immuno-incompetent or immuno-compromised.

The present invention is generally applied to humans. In certainembodiments, non-human mammals, such as mice and rats, may also be usedfor the purpose of demonstration. One may use the present invention forveterinary purpose. For example, one may wish to treat commerciallyimportant farm animals, such as cows, horses, pigs, rabbits, goats, andsheep. One may also wish to treat companion animals, such as cats anddogs.

Adjuvants of the Present Invention

As used herein, “Th17 cells” refers to a population of CD4+ T cellswhich produce Il-17. As used herein, “Th1 cells” refers to a populationof CD4+ T cells which produce INF-γ. As used herein, “Tc1 cells” refersto a population of cytotoxic CD8+ T cells that produce INF-γ. Vaccineinduced CD4+ T cells that produce IL-17 (Th17 cells) and INF-γ (Th1cells) and CD8+Tc1 cells that produce INF-γ are active in resistanceagainst fungal, bacterial and viral infections. In one embodiment of theinvention, the vaccine requires the activity of the Dectin-2 receptor onphagocytes that will trigger the development of antigen-specific Th17and Th1 cells to mediate resistance.

As used herein, the term “Dectin-2” or “Dec-2” refers to a type IItransmembrane C-type lectin receptor involved in the innate immuneresponse. Note to be bound by any particular theory, Applicants workingtheory indicates that fungal vaccine recognition by theDectin-2/FcRγ/Syk/Card9 signaling axis is required for thedifferentiation of Th17 and Th1 cells and the induction ofvaccine-induced resistance to fungal infection (Wang et. al 2014 JImmunol).

As used herein, the term “Dectin-2 ligand” refers to a molecule capableof binding to or activating the Dectin-2/FcRγ/Syk/Card9 signaling axisto promote the differentiation of Th17 and Th1 cells. The molecule maybe a protein, a lipid, a glycoprotein, a glycolipid or any glycancapable of binding Dectin-2. A suitable Dectin-2 ligand of the presentinvention is characterized by the ability to induce Dectin-2 signalingusing Dectin-2 expressing B3Z T cell reporter cells (FIG. 2C) or toproduce cytokines by bone marrow derived dendritic cells (BMDC) in aDectin-2 dependent manner (for example, when comparing cytokineproduction by wild type vs. Dectin-2-deficient BMDCs) (FIG. 2D+E) andincreasing the activation and differentiation of antigen-specific Tcells in vivo (FIG. 3). In one embodiment the Dectin-2 ligand of thepresent invention is selected from the group consisting of Bl-Eng2,MP98, Furfurman from Malassezia sp., and Man-LAM (Ishikawa et al. 2003,Yonekawa et al. 2014). In another embodiment of the invention, theDectin-2 ligand is a glycoprotein selected from the group consisting ofMP98 and Bl-Eng2.

As used herein, the term “Bl-Eng2” refers to the fungal glycoproteinβ-1,3-endoglucansase from Blastomyces dermatitidis. Bl-Eng2 has homologyto Aspergillus fumigates endoglucanase 2 (Eng2) at the C-terminalglycosylation site, and endoglucanase 3 (Eng3) at the active site.

The predicted molecular weight of Bl-Eng2, based on amino acid sequencealone, is 57 kDa. However, Bl-Eng2 may appear as a 115-130 kDa band onan SDS-PAGE gel based on post-translation glycosylation. Bl-Eng2comprises an 18 amino acid signal peptide, an N-terminal GH16 glycosylhydrolase catalytic domain, and a C-terminal S/T-rich domain. Bl-Eng2undergoes post-translational modification and has a number of O-linkedglycosylation sites, which may be in the S/T-rich C-terminal domain. Itis understood that mannose is the major monosaccharide present in thePTM glycosylation of Bl-Eng2 when it is expressed in Pichia pastoris. Inone embodiment B1-Eng2 comprises the sequence of SEQ ID NO: 1. In oneembodiment, the Bl-Eng2 is a fragment, single domain, or short glycanfragment of the full-length Bl-Eng2.

As used herein, the term “MP98” refers to the chitin deacetylase-likeprotein from Cryptococcus neoformans (Levitz et al., 2001). MP98comprises an N-terminal cleavable signal sequence, a polysaccharidedeacetylase domain found in fungal chitin deacetylases, and aserine/threonine-rich C-terminal region. The C-terminal region comprisesN-liked glycosylation sites comprises covalently linked mannose.

A Dectin-2 ligand suitable for use as a vaccine adjuvant in the presentinvention may be in any form as discussed above. In one embodiment, theDectin-2 ligand may be expressed in commercially available sources,e.g., Pichia pastoris. The Dectin-2 ligand maybe expressed in anycommercially available sources that is capable of post-translationalprotein modifications. The Dectin-2 ligand vaccine adjuvant may be thenisolated and purified from these sources. The protein expression,isolation, and purifications are well known to a person having ordinaryskill in the art. The Examples demonstrate methods of expression,isolation, and purifications of Bl-Eng2 according to one embodiment ofthe present invention.

A vaccine comprising a Dectin-2 ligand adjuvant may also comprise othersuitable ingredients. In one embodiment, a vaccine may also comprise acarrier molecule as a stabilizer component. As the types of vaccinesenclosed in the present invention may be rapidly degraded once injectedinto the body, the vaccine may be bound to a carrier molecule forstabilizing the vaccine during delivery and administration. A suitablecarrier or stabilizer may comprise fusion proteins, polymers, liposomes,micro- or nanoparticles, or any other pharmaceutically acceptablecarriers. A suitable carrier or stabilizer molecule may comprise atertiary amine N-oxide, e.g., trimethylamine-N-oxide, a sugar, e.g.,trehalose, a poly(ethylene glycol) (PEG), an animal-based protein, e.g.,digested protein extracts such as N-Z-Amine®, N-Z-Amine AS® andN-Z-Amine YT® (Sheffield Products Co., Norwich, N.Y.), a vegetable-basedprotein, e.g., soy protein, wheat protein, corn gluten, rice protein andhemp protein, and any other suitable carrier molecules.

As used herein “glucan particle” refers to a formulation of the vaccinecomprising β_(1,3) glucan particles as a solid support. Glucan particlestarget Dectin-1, a key pattern recognition receptor for anti-fungalimmunity. Glucan particles also serve as structural vessels or a type orstructural scaffold to deliver antigen as well as adjuvants in thevaccine formulation. In one embodiment, β_(1,3) glucan particles (GPs)are used as a solid support for Bl-Eng2. In one embodiment, glucanparticles in used in a vaccine formulation with calnexin fragments and aDectin-2 ligand adjuvant against pathogenic fungi.

In another aspect, the Dectin-2 ligand adjuvant of the present inventionmay be administered in a formulation with any known commerciallyavailable adjuvant in the art. Adjuvants to be administrated mayinclude, but are not limited to, aluminum hydroxide (Alum), glucanparticles engaging Dectin-1, Adjuplex, and combinations thereof. It isalso envisioned that when Bl-Eng3 is administered in a vaccinecomposition as an antigen, it may be administrated with a suitableadjuvant. Suitable adjuvants are any commercially available adjuvantknown in the art or an adjuvant described herein.

Suitable Carrier or Vehicle

Suitable agents may include a suitable carrier or vehicle for delivery.As used herein, the term “carrier” refers to a pharmaceuticallyacceptable solid or liquid filler, diluent or encapsulating material. Awater-containing liquid carrier can contain pharmaceutically acceptableadditives such as acidifying agents, alkalizing agents, antimicrobialpreservatives, antioxidants, buffering agents, chelating agents,complexing agents, solubilizing agents, humectants, solvents, suspendingand/or viscosity-increasing agents, tonicity agents, wetting agents orother biocompatible materials. A tabulation of ingredients listed by theabove categories, may be found in the U.S. Pharmacopeia NationalFormulary, 1857-1859, (1990).

Some examples of the materials which can serve as pharmaceuticallyacceptable carriers are sugars, such as lactose, glucose and sucrose;starches such as corn starch and potato starch; cellulose and itsderivatives such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipientssuch as cocoa butter and suppository waxes; oils such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; glycols, such as propylene glycol; polyols such asglycerin, sorbitol, mannitol and polyethylene glycol; esters such asethyl oleate and ethyl laurate; agar; buffering agents such as magnesiumhydroxide and aluminum hydroxide; alginic acid; pyrogen free water;isotonic saline; Ringer's solution, ethyl alcohol and phosphate buffersolutions, as well as other nontoxic compatible substances used inpharmaceutical formulations. Wetting agents, emulsifiers and lubricantssuch as sodium lauryl sulfate and magnesium stearate, as well ascoloring agents, release agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be presentin the compositions, according to the desires of the formulator.

Examples of pharmaceutically acceptable antioxidants include watersoluble antioxidants such as ascorbic acid, cysteine hydrochloride,sodium bisulfite, sodium metabisulfite, sodium sulfite and the like;oil-soluble antioxidants such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol and the like; and metal-chelating agents suchas citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid and the like.

Stabilization Agent

In another configuration, the present formulation may also compriseother suitable agents that stabilize the formulations. For example, anapproach for stabilizing solid protein formulations of the invention isto increase the physical stability of purified, e.g., lyophilized,protein. This will inhibit aggregation via hydrophobic interactions aswell as via covalent pathways that may increase as proteins unfold.Stabilizing formulations in this context may often include polymer-basedformulations, for example a biodegradable hydrogel formulation/deliverysystem. The critical role of water in protein structure, function, andstability is well known. Typically, proteins are relatively stable inthe solid state with bulk water removed. However, solid therapeuticprotein formulations may become hydrated upon storage at elevatedhumidities or during delivery from a sustained release composition ordevice. The stability of proteins generally drops with increasinghydration. Water may also play a significant role in solid proteinaggregation, for example, by increasing protein flexibility resulting inenhanced accessibility of reactive groups, by providing a mobile phasefor reactants, and by serving as a reactant in several deleteriousprocesses such as beta-elimination and hydrolysis.

An effective method for stabilizing peptides and proteins againstsolid-state aggregation for delivery may be to control the water contentin a solid formulation and maintain the water activity in theformulation at optimal levels. This level depends on the nature of theprotein, but in general, proteins maintained below their “monolayer”water coverage will exhibit superior solid-state stability.

A variety of additives, diluents, bases and delivery vehicles may beprovided within the invention that effectively control water content toenhance protein stability. These reagents and carrier materialseffective as anti-aggregation agents in this sense may include, forexample, polymers of various functionalities, such as polyethyleneglycol, dextran, diethylaminoethyl dextran, and carboxymethyl cellulose,which significantly increase the stability and reduce the solid-phaseaggregation of peptides and proteins admixed therewith or linkedthereto. In some instances, the activity or physical stability ofproteins may also be enhanced by various additives to aqueous solutionsof the peptide or protein drugs. For example, additives, such as polyols(including sugars), amino acids, proteins such as collagen and gelatin,and various salts may be used.

Certain additives, in particular sugars and other polyols, may alsoimpart significant physical stability to dry, e.g., lyophilizedproteins. These additives may also be used within the invention toprotect the proteins against aggregation not only during lyophilizationbut also during storage in the dry state. For example sucrose and Ficoll70 (a polymer with sucrose units) exhibit significant protection againstpeptide or protein aggregation during solid-phase incubation undervarious conditions. These additives may also enhance the stability ofsolid proteins embedded within polymer matrices.

Yet additional additives, for example sucrose, stabilize proteinsagainst solid-state aggregation in humid atmospheres at elevatedtemperatures, as may occur in certain sustained-release formulations ofthe invention. Proteins such as gelatin and collagen also serve asstabilizing or bulking agents to reduce denaturation and aggregation ofunstable proteins in this context. These additives can be incorporatedinto polymeric melt processes and compositions within the invention. Forexample, polypeptide microparticles can be prepared by simplylyophilizing or spray drying a solution containing various stabilizingadditives described above. Sustained release of unaggregated peptidesand proteins can thereby be obtained over an extended period of time.

Various additional preparative components and methods, as well asspecific formulation additives, are provided herein which yieldformulations for mucosal delivery of aggregation-prone peptides andproteins, wherein the peptide or protein is stabilized in asubstantially pure, unaggregated form using a solubilization agent. Arange of components and additives are contemplated for use within thesemethods and formulations. Exemplary of these solubilization agents arecyclodextrins (CDs), which selectively bind hydrophobic side chains ofpolypeptides. These CDs have been found to bind to hydrophobic patchesof proteins in a manner that significantly inhibits aggregation. Thisinhibition is selective with respect to both the CD and the proteininvolved. Such selective inhibition of protein aggregation may provideadditional advantages within the intranasal delivery methods andcompositions of the invention.

Additional agents for use in this context include CD dimers, trimers andtetramers with varying geometries controlled by the linkers thatspecifically block aggregation of peptides and protein. Yetsolubilization agents and methods for incorporation within the inventioninvolve the use of peptides and peptide mimetics to selectively blockprotein-protein interactions. In one aspect, the specific binding ofhydrophobic side chains reported for CD multimers may be extended toproteins via the use of peptides and peptide mimetics that similarlyblock protein aggregation. A wide range of suitable methods andanti-aggregation agents may be available for incorporation within thecompositions and procedures of the invention.

Stabilizing Delivery Vehicle, Carrier, Support or Complex-FormingSpecies

In another embodiment, the present formulation may also comprise othersuitable agents such as a stabilizing delivery vehicle, carrier, supportor complex-forming species. The coordinate administration methods andcombinatorial formulations of the instant invention may optionallyincorporate effective lipid or fatty acid based carriers, processingagents, or delivery vehicles, to provide improved formulations fordelivery of Dectin-2 ligand or functionally equivalent fragmentproteins, analogs and mimetics, and other biologically active agents andantigens of the composition. For example, a variety of formulations andmethods are provided for delivery which comprise one or more activeagents, including the Dectin-2 ligand adjuvant, such as a peptide orprotein, admixed or encapsulated by, or coordinately administered with,a liposome, mixed micellar carrier, or emulsion, to enhance chemical andphysical stability and increase the half-life of the biologically activeagents (e.g., by reducing susceptibility to proteolysis, chemicalmodification and/or denaturation) upon mucosal delivery.

Within certain aspects of the invention, specialized delivery systemsfor biologically active agents may comprise small lipid vesicles knownas liposomes or micelles. These are typically made from natural,biodegradable, non-toxic, and non-immunogenic lipid molecules, and canefficiently entrap or bind drug molecules, including peptides andproteins, into, or onto, their membranes. The attractiveness ofliposomes as a peptide and protein delivery system within the inventionis increased by the fact that the encapsulated proteins can remain intheir preferred aqueous environment within the vesicles, while theliposomal membrane protects them against proteolysis and otherdestabilizing factors. Even though not all liposome preparation methodsknown are feasible in the encapsulation of peptides and proteins due totheir unique physical and chemical properties, several methods allow theencapsulation of these macromolecules without substantial deactivation.

Additional delivery vehicles carrier, support or complex-forming speciesfor use within the invention may include long and medium chain fattyacids, as well as surfactant mixed micelles with fatty acids. Mostnaturally occurring lipids in the form of esters have importantimplications with regard to their own transport across mucosal surfaces.Free fatty acids and their monoglycerides which have polar groupsattached have been demonstrated in the form of mixed micelles to act onthe intestinal barrier as penetration enhancers. This discovery ofbarrier modifying function of free fatty acids (carboxylic acids with achain length varying from 12 to 20 carbon atoms) and their polarderivatives has stimulated extensive research on the application ofthese agents as mucosal absorption enhancers.

For use within the methods of the invention, long chain fatty acids,especially fusogenic lipids (unsaturated fatty acids and monoglyceridessuch as oleic acid, linoleic acid, linoleic acid, monoolein, etc.)provide useful carriers to enhance delivery of Calnexin or afunctionally equivalent fragment, and other biologically active agentsdisclosed herein. Medium chain fatty acids (C6 to C12) andmonoglycerides have also been shown to have enhancing activity inintestinal drug absorption and can be adapted for use within the mucosaldelivery formulations and methods of the invention. In addition, sodiumsalts of medium and long chain fatty acids are effective deliveryvehicles and absorption-enhancing agents for mucosal delivery ofbiologically active agents within the invention. Thus, fatty acids canbe employed in soluble forms of sodium salts or by the addition ofnon-toxic surfactants, e.g., polyoxyethylated hydrogenated castor oil,sodium taurocholate, etc. Other fatty acid and mixed micellarpreparations that are useful within the invention include, but are notlimited to, Na caprylate (C8), Na caprate (C10), Na laurate (C12) or Naoleate (C18), optionally combined with bile salts, such as glycocholateand taurocholate.

The vaccine formulation may additionally include a biologicallyacceptable buffer to maintain a pH close to neutral (7.0-7.3). Suchbuffers preferably used are typically phosphates, carboxylates, andbicarbonates. More preferred buffering agents are sodium phosphate,potassium phosphate, sodium citrate, calcium lactate, sodium succinate,sodium glutamate, sodium bicarbonate, and potassium bicarbonate. Thebuffer may comprise about 0.0001-5% (w/v) of the vaccine formulation,more preferably about 0.001-1% (w/v). The buffer(s) may be added as partof the stabilizer component during the preparation thereof, if desired.Other excipients, if desired, may be included as part of the finalvaccine formulation.

The remainder of the vaccine formulation may be an acceptable diluent,to 100%, including water. The vaccine formulation may also be formulatedas part of a water-in-oil, or oil-in-water emulsion.

Also provided as part of the invention is a method of preparation of thevaccine formulation described herein. Preparation of the vaccineformulation preferably takes place in two phases. The first phase maytypically involve the preparation of the stabilizer component. Thestabilizer component may comprise any suitable components as discussedabove. For example, a vegetable-based protein stock solution may beprepared by dissolving the vegetable-based protein in a diluent. Thepreferred diluent may be water, preferably distilled and/or purified soas to remove trace impurities (such as that sold as purified Super Q®).In a separate vessel an animal-based protein may be dissolved in adiluent, additionally with the sugar component and buffer additives.Preferably, an equal volume of the vegetable-based protein stocksolution is added to the animal-based protein solution. It is desirablethat after HCl/KOH adjustment to achieve a pH of approximately 7.2±0.1,the stabilizer component may be sterilized via autoclave. The stabilizersolution may be refrigerated for an extended period prior tointroduction of the Dectin-2 ligand adjuvant and a suitable antigen.

The second phase of preparation of the vaccine formulation may includeintroduction of the Dectin-2 ligand adjuvant and a suitable antigen withthe stabilizer component, thereby yielding the vaccine formulation.Preferably, the Dectin-2 ligand adjuvant may be diluted with a buffersolution prior to its introduction to the stabilizer component.

Once this vaccine formulation solution has been achieved, theformulation may be separated into vials or other suitable containers.The vaccine formulation herein described may then be packaged inindividual or multi-dose ampoules, or be subsequently lyophilized(freeze-dried) before packaging in individual or multi-dose ampoules.The vaccine formulation herein contemplated also includes thelyophilized version. The lyophilized vaccine formulation may be storedfor extended periods of time without loss of viability at ambienttemperatures. The lyophilized vaccine may be reconstituted by the enduser, and administered to a patient.

The term “lyophilization” or “lyophilized,” as used herein, refers tofreezing of a material at low temperature followed by dehydration bysublimation, usually under a high vacuum. Lyophilization is also knownas freeze drying. Many techniques of freezing are known in the art oflyophilization such as tray-freezing, shelf-freezing, spray-freezing,shell-freezing and liquid nitrogen immersion. Each technique will resultin a different rate of freezing. Shell-freezing may be automated ormanual. For example, flasks can be automatically rotated by motor drivenrollers in a refrigerated bath containing alcohol, acetone, liquidnitrogen, or any other appropriate fluid. A thin coating of product isevenly frozen around the inside “shell” of a flask, permitting a greatervolume of material to be safely processed during each freeze drying run.Tray-freezing may be performed by, for example, placing the samples inlyophilizer, equilibrating 1 hr at a shelf temperature of 0° C., thencooling the shelves at 0.5° C./min to −40° C. Spray-freezing, forexample, may be performed by spray-freezing into liquid, dropping by ˜20μl droplets into liquid N₂, spray-freezing into vapor over liquid, or byother techniques known in the art.

The vaccine of the present invention may be either in a solid form or ina liquid form. Preferably, the vaccine of the present invention may bein a liquid form. The liquid form of the vaccine may have aconcentration of 50-4,000 nanomolar (nM), preferably between 50-150 nM.In some embodiments, the concentration will be between 1-50,000 nM.

To vaccinate a patient, a therapeutically effective amount of vaccinecomprising a suitable antigen and a Dectin-2 ligand adjuvant may beadministered to a patient. The therapeutically effective amount ofvaccine may typically be one or more doses, preferably in the range ofabout 0.01-10 mL, most preferably 0.1-1 mL, containing 1-200 micrograms,most preferably 1-100 micrograms of vaccine formulation/dose. Thetherapeutically effective amount may also depend on the vaccinationspecies. For example, for smaller animals such as mice, a preferreddosage may be about 0.01-1 mL of a 1-50 microgram solution of antigen.For a human patient, a preferred dosage may be about 0.1-1 mL of a 1-50microgram solution of antigen. The therapeutically effective amount mayalso depend on other conditions including characteristics of the patient(age, body weight, gender, health condition, etc.), the species offungi, and others. In one embodiment the vaccine formulation of thepresent invention comprises 1-100 micrograms of Dectin-2 ligand adjuvantand 5-20 micrograms of Calnexin fragment in either soluble or glucanparticle formulation.

In another aspect, to vaccinate a patient against a fungal infection, atherapeutically effective amount of a vaccine comprising Bl-Eng2 as anantigen may be administered to a patient. The therapeutically effectiveamount of vaccine my typically be one or more doses, preferably in therange of about 0.01-10 mL, most preferably 0.1-1 mL, containing 1-200micrograms, most preferably 1-100 micrograms of vaccineformulation/dose. The vaccine my comprise 1-100 micrograms of Bl-Eng2 asan antigen.

A vaccine of the present invention may be administered by using anysuitable means as disclosed above. Preferably, a vaccine of the presentinvention may be administered by intranasal delivery, transmucosaladministration, subcutaneous or intramuscular administration, e.g.,needle injection. In some embodiments, vaccine compositions forprotection against a viral infection are formulated for transmucosaldelivery. In some embodiments, vaccine compositions for protectionagainst a bacterial infection are formulated for subcutaneousadministration.

After vaccination using a vaccine of the present invention comprisingthe Dectin-2 ligand adjuvant, a patient may be immunized against atleast one type of fungi, bacteria, or virus. In one specific embodiment,a patient after vaccination may be immunized against at least onespecies of dimorphic fungi. In one preferred embodiment, a patient aftervaccination may be immunized from multiple dimorphic fungi includingHistoplasma, Coccidiodes, Paracoccidioides, Penicillium, Blastomyces,Sporothrix, and Aspergillus fumigatus

The instant invention may also include kits, packages and multicontainerunits containing the above described pharmaceutical compositions, activeingredients, and/or means for administering the same for use in theprevention and treatment of diseases and other conditions in mammaliansubjects. Briefly, these kits include a container or formulation thatcontains the Bl-Eng2 adjuvant or a functionally equivalent fragment,and/or other biologically active agents in combination with mucosal orsubcutaneous delivery enhancing agents disclosed herein formulated in apharmaceutical preparation for delivery.

As used herein, the term “pharmaceutically acceptable carrier” refers toany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and the likethat are physiologically compatible. Preferably, the carrier is suitablefor intravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the antigentic peptide, i.e., the calnexinprotein may be coated in a material to protect the peptide from theaction of acids and other natural conditions that may inactivate thepeptide.

A “pharmaceutically acceptable salt” refers to a salt that retains thedesired biological activity of the parent compound and does not impartany undesired toxicological effects (see e.g., Berge, S. M., et al.(1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acidaddition salts and base addition salts. Acid addition salts includethose derived from nontoxic inorganic acids, such as hydrochloric,nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous andthe like, as well as from nontoxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acidsand the like. Base addition salts include those derived from alkalineearth metals, such as sodium, potassium, magnesium, calcium and thelike, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

The composition can be formulated as a solution, microemulsion,dispersion, liposome, or other ordered structure suitable to high drugconcentration.

In one embodiment, the composition may also comprise a carrier moleculeas a stabilizer component. As the types of proteins or peptides enclosedin the present invention may be rapidly degraded once injected into thebody, the proteins or peptides may be bound to a carrier molecule forstabilizing the proteins or peptides during delivery and administration.A suitable carrier or stabilizer may comprise fusion proteins, polymers,liposome, micro or nanoparticles, or any other pharmaceuticallyacceptable carriers. A suitable carrier or stabilizer molecule maycomprise a tertiary amine N-oxide, e.g., trimethylamine-N-oxide, asugar, e.g., trehalose, a poly(ethylene glycol) (PEG), an animal-basedprotein, e.g., digested protein extracts such as N-Z-Amine®, N-Z-AmineAS® and N-Z-Amine YT® (Sheffield Products Co., Norwich, N.Y.), avegetable-based protein, e.g., soy protein, wheat protein, corn gluten,rice protein and hemp protein, and any other suitable carrier molecules.The composition may also comprise any suitable carrier or vehicle, suchas those as discussed above. The composition may also comprise otherstabilization agents, such as those as discussed above.

In one embodiment, the composition may also comprise suitablestabilizing delivery vehicle, carrier, support or complex-formingspecies, such as those as discussed above. For example, the compositionmay additionally comprise at least one of a stabilizer, a buffer, or anadjuvant.

The present invention has been described in terms of one or morepreferred embodiments, and it should be appreciated that manyequivalents, alternatives, variations, and modifications, aside fromthose expressly stated, are possible and within the scope of theinvention.

Example 1

Adaptive immunity is critical for the prevention and resolution offungal infections. The contribution of antibodies to host defense isdebated. In contrast, Ag-specific CD4⁺ T cells play the major role infungal resistance, as evidenced by the high incidence oflife-threatening fungal infections in patients with impaired CD4⁺ Tcells. CD4⁺ T cells confer resistance by secretion of T-helper 1 (Th1)and Th17 cytokines such as IFN-γ, TNF-α, GM-CSF, and IL-17A, whichactivate neutrophils, monocytes, macrophages and DCs for fungalclearance. Since CD4⁺ T cells are germane to host defense against fungi,the challenge is how best to elicit these T cells.

The transition from innate to adaptive immunity is fostered by dendriticcells (DCs). These cells process and present Ag to naïve CD4⁺ T cells inthe context of co-stimulatory factors (e.g. cell surface ligands andcytokines) that provide the combination of signals necessary to inducenaive T cell activation and proliferation. During their interactionswith DCs, naive T cells also become functionally specialized. Helper Tcell polarization occurs as a result of the cytokines produced by DCs:Th1 polarization is associated with DC production of high levels ofIL-12p70, and Th17 polarization is associated with DC production ofIL-1β and IL-6. While vaccine Ags typically have little impact on thenature of the cytokines produced by DCs, the adjuvant can have adramatic effect. Alum (aluminum hydroxide), which is the most commonlyused adjuvant in current vaccine formulations, suppresses DC productionof pro-inflammatory cytokines such as IL-12p70, creating an environmentthat polarizes T cells towards a Th2 phenotype. Thus, a major weaknessand central challenge in the field of vaccinology is the lack ofadjuvants that drive Th1 and/or Th17 polarization and stimulate DCs toproduce the appropriate cytokines. Pathways that can differentiallyactivate DC cytokine profiles include toll-like receptors (TLRs), C-typelectin receptors (CLRs), co-stimulatory ligands such as CD40, andcytokine receptors.

C-type lectins are important in fungal recognition by DCs and ininducing anti-fungal Th1 and Th17 responses. Dectin-1 and Dectin-2induce Th1/Th17 cells in response to Candida albicans and Aspergillusfumigatus infection. While Dectin-1 is dispensable, Dectin-2 isrequisite for the development of protective Th1 and Th17 cells andvaccine resistance against dimorphic fungi. Crude fractions ofmannoproteins isolated from Malassezia pachydermatis as well as alipoglycan (Man-LAM) of Mycobacterium tuberculosis have been shown totrigger Dectin-2 signaling, however they have not been evaluated asvaccine adjuvants, and glycans and lipids may be difficult to expressand scale.

The embodiment described here demonstrates a novel fungal Dectin-2ligand from an attenuated vaccine strain of Blastomyces dermatitidis,Bl-Eng2. We tested whether the ligation of Dectin-2 effectivelyvaccinates mice against fungi. Our vaccination strategy was to ligateDectin-2 with Bl-Eng2 and assess the adjuvant activity by combining itwith the recently reported pan-fungal vaccine calnexin. Fungalrecombinant Bl-Eng2 was expressed and scaled efficiently, it stimulatedIL-6 and IL-1β production in vitro and Th1 and Th17 cells in vivo and,when used as an adjuvant in combination with calnexin, it protected miceagainst pneumonia in a model of lethal pulmonary fungal infection.

Results

B. dermatitidis vaccine yeast are bound by soluble Dectin-2 fusionprotein and trigger NFAT signaling of Dectin-2 reporter cells.Dectin-2^(−/−) mice fail to develop Ag-specific Th1 and Th17 cells oracquire vaccine resistance. We therefore sought to identify the fungalpathogen-associated molecular pattern (PAMP) that is recognized byDectin-2. We used the NFAT-LacZ reporter cells to enrich Dectin-2 ligandactivity from the vaccine yeast cell wall. We sonicated vaccine yeast,collected the water-soluble, cell-wall extract (CWE) and analyzed it bySDS-PAGE. CWE displayed a broad range of protein bands (FIG. 1A) andharbored Dectin-2 ligand activity (FIG. 1B). Digestion of CWE withproteinase K or endo-mannosidases reduced this activity (FIG. 1B),suggesting that both protein and glycan moieties may contribute toligand activity. To define the Mr of candidate proteins, we separatedthe CWE using a GELFREE 8100 system (FIG. 6A). Fractions #5-6 rangingbetween 75 to 150 kDa in size contained ligand activity (FIGS. 6A-6B).

To enrich and identify glycoprotein with ligand activity, we employedthe lectin Concanavalin A (ConA), which binds α-D-mannose andα-D-glucose moieties, gel filtration and Mass spectrometry analysis(FIG. 1C). The majority of the ligand activity was removed from CWE by aConA resin, and the eluate was highly enriched for ligand activity(FIGS. 1D-1E). The enrichment by ConA suggested that the Dectin-2ligand(s) in CWE are mannoproteins. To further enrich ligand activity,the ConA eluate was separated by size exclusion chromatography twice,sequentially. Fractions F4-F6 after the first run contained ligandactivity as determined by the Dectin-2 reporter assay (FIG. 1F); F4-6were pooled and subjected to a second separation by gel filtration. Thepositive fractions (F9-F13) and negative ones (F1-F7) after the secondgel filtration (FIG. 6C) were analyzed by mass spectrometry (FIGS. 2Aand 7A). Proteins that were more abundant in the positive vs. negativefraction were considered candidates. Among the candidates, anuncharacterized member (BDFG 08749) of the fungalendo-1,3(4)-β-D-glucanase family stood out in positive fractions (FIG.7A). The native 526-aa protein contains an 18-aa signal peptide, anN-terminal GH16 glycosyl hydrolase (GH) catalytic domain, and aC-terminal S/T-rich domain (FIGS. 2B and 7B) that could be responsiblefor the strong glycosylation (FIGS. 2C-2D). The GH16 catalytic domain ofthe endo glucanase has 60.1% similarity (identical aa and conservativesubstitution) (45.8% identity) and the entire glycoprotein has 45.2%similarity (28.8% identity) to the GPI-anchored endo β-1,3-glucanaseEng2 of A. fumigatus. Thus, we named the protein ligand Blastomyces-Eng2(B1-Eng2). PDIA1 is a protein that was more abundant in the negative gelfiltration fraction (e.g. a negative control) (FIG. 2A) and showed noreporter activity and little glycosylation (FIG. 2C).

Bl-Eng2 Protein is a Bona-Fide Ligand for Dectin-2—

To evaluate whether Bl-Eng2 is recognized by Dectin-2, we cloned andexpressed the recombinant protein in Pichia pastoris. This eukaryoticexpression system modifies recombinant proteins with both O- andN-linked glycosylation. Full-length Bl-Eng2 was fused to a N-terminalα-factor secretion signal and a C-terminal Myc-6×His tag (FIG. 2B).Ni-NTA purified Bl-Eng2 showed a band of ˜120 kDa on SDS-PAGE gel (FIG.2C), which falls within the size range determined in FIG. 6A+B. Periodicacid-Schiff (PAS) based glyco-stain of Bl-Eng2 showed strongglycosylation (FIG. 2C), which likely accounts for the discrepancybetween predicted Mr of 57 kDa and apparent Mr of ˜120 kDa. Gaschromatography (GC) analysis indicated that mannose is the majormonosaccharide, and constitutes 82.8% in glycan mass of Pichia-expressedBl-Eng2 (FIG. 2D).

To verify Bl-Eng2 ligand activity, B3Z reporter cells expressingDectin-2 or other distinct CLRs were incubated with recombinant Bl-Eng2.Bl-Eng2 elicited strong NFAT-lacZ signalling from Dectin-2 reportercells, but not from the other CLR-expressing cells (FIG. 3A), indicatinga specific interaction between Dectin-2 and Bl-Eng2. Since AspergillusEng2 (Asp-Eng2) exhibits a high degree of similarity to Bl-Eng2 andcontains a Ser/Thr-rich C terminus, we also tested whether Asp-Eng2 isrecognized by Dectin-2. Asp-Eng2 and Bl-Eng2 were similarly recognizedby Dectin-2 expressing reporter cells (FIGS. 8A-8B), hence Eng2 fromboth fungal species are Dectin-2 ligands.

Dectin-2 is required for Bl-Eng2 ligand activity in primary cells—Toinvestigate whether Bl-Eng2 stimulates primary cells, we examinedpro-inflammatory cytokine production from bone marrow-derived dendriticcells (BMDCs). BMDCs from wild type mice, but not Dectin-2^(−/−) orCard9^(−/−) mice, produced a strong IL-6 response when stimulated withrecombinant Bl-Eng2, but not PDIA1 (FIG. 3B), indicating ligandspecificity for Dectin-2. Lack of stimulation of BMDCs from knockoutmice also excludes the possibility of endotoxin contamination as thestimulus of IL-6 in wild type cells. Thus, Pichia-expressed Bl-Eng2triggers a cytokine response in vitro that requires Dectin-2 anddownstream Card9. These results together indicate that Bl-Eng2 appearsto be a selective Dectin-2 ligand.

Bl-Eng2 is a Dectin-2 Ligand with Superior Capacity to Elicit CytokineResponses—

Dectin-2 recognizes several fungi including C. albicans, A. fumigatusand Malassezia, which possess N- and O-linked mannan on their surface.Thus, not surprisingly, there are two other Dectin-2 ligands describedin the literature. They are Furfurman from Malassezia spp. and Man-LAMfrom M. tuberculosis. In addition to these ligands, by using B3Zreporter cells in the work here, we observed that MP98 from Cryptococcusneoformans is also recognized by Dectin-2 (FIG. 8C). MP98 also triggersIL-6 by BMDC in a Dectin-2- and concentration-dependent manner (FIG.8D). MP98 is a mannoprotein of Mr of 98 kDa with 103 Ser/Thr residues atthe C-terminus that serve as potential O-linked glycosylation sites, and12 putative N-linked glycosylation sites.

To begin to evaluate the relative potency of Dectin-2 ligands, wecompared the ability of Bl-Eng2 and the other three Dectin-2 ligands toinduce cytokine production by BMDCs. Bl-Eng2 induced the strongest IL-6production by BMDCs when compared at equal molar and mass ratios to theother ligands (FIG. 3C). These results suggest that Bl-Eng2 isrelatively potent for triggering IL-6 and might be used as an adjuvantfor vaccination to boost the development of Ag-specific T cellresponses.

Bl-Eng2 Induces the Production of IL-6 and IL-1,8 by Human PBMCs—

A suitable adjuvant for vaccine formulation should ideally stimulatehuman accessory cells. To test this capacity, we assessed the effect ofBl-Eng2 on the function of human PBMCs. After stimulation withplate-coated Bl-Eng2, human PBMCs from five healthy subjects produced upto 17 ng/ml IL-6 and 9 ng/ml IL-1β as measured in the cell culturesupernatants by ELISA (FIG. 3D). These data suggest that recombinantBl-Eng2 has the capacity to induce the production of Th17 cell primingcytokines by human antigen-presenting cells (APC) in vitro.

Bl-Eng2 Promotes T Cell Development In Vivo and Imparts VaccineEfficacy—

To investigate whether Bl-Eng2 could be harnessed as a vaccine adjuvant,we performed preclinical studies in mice. We first tested whetherBl-Eng2 augments the development of vaccine Ag-specific T cells. Toassess these T cell responses in vivo, we vaccinated mice with thepan-fungal Ag calnexin and enumerated CD4⁺ T cell responses by TCR Tg1807 cells, which are specific for calnexin. Calnexin was suspended withincomplete freund's adjuvant (mineral oil) and injected subcutaneously.The addition of Bl-Eng2 into the formulation sharply increased thefrequency of IL-17 producing 1807 T cells (FIG. 4A) and the number ofactivated (CD44⁺) and IL-17 and IFN-γ producing 1807 T cells, asmeasured by ex vivo stimulation with anti-CD3 and anti-CD28 mAb (FIGS.4B and 9A). Ex vivo stimulation with the vaccine Ag calnexin alsoyielded sharp increases in the amount of IL-17 produced by T cells fromthe draining lymph nodes (FIG. 4D). Thus, Bl-Eng2 promoted thedevelopment of Th17 cells more so than Th1 cells.

Addition of Bl-Eng2 to the vaccine also reduced lung CFU as early asfour days after mice received a lethal experimental challenge, and didso in a concentration-dependent manner (FIG. 9B). In a parallel group,at the time unvaccinated control mice were moribund (day 18post-infection), the addition of Bl-Eng2 to the vaccine reduced lung CFUby more than two logs (FIG. 4C). Combining the vaccine with commercialalum as an adjuvant did not increase the frequency and numbers ofcytokine producing T cells or reduce the fungal burden (FIGS. 4A-4D).However, combining Bl-Eng-2 together with Alum increased the adjuvancyof Alum as measured by the number of activated (CD44⁺), IL-17 and IFN-γproducing 1807 T cells and the reduction in lung CFU (FIG. 10). Theseresults suggest that Bl-Eng-2 can work in concert with other(commercially available and FDA approved) adjuvants and augment vaccineefficacy.

Bl-Eng2 failed to increase the development of Th17 and Th1 cells, theproduction ex vivo of IL-17 and IFN-γ or reduce lung CFU inDectin-2^(−/−) mice, verifying that the adjuvant effect isDectin-2-dependent in vivo (FIGS. 9C-9E). Thus, Bl-Eng2 exhibitsadjuvant-like properties by increasing the development of Ag-specific(1807) Th17 and Th1 cells and protecting mice from lethal pulmonaryinfection with B. dermatitidis.

The studies above exploited TCR Tg T cells to sensitively report theability of Bl-Eng2 to enhance development of calnexin Ag-specific Th17and Th1 cells upon vaccination. However, adoptive transfer of thesecells into mice artificially enhances the number of CD4⁺ T cellprecursors in the animal. To investigate whether Bl-Eng2 also has thecapacity to induce the development of endogenous calnexin-Ag specificCD4⁺ T cells and similarly protect animals, we vaccinated wild type micein the absence of adoptive transfer. The formulation of Bl-Eng2 with thecalnexin subunit vaccine again reduced lung CFU by over two logs vs.control mice vaccinated with calnexin in mineral oil alone (IFA), and byover 3 logs vs. mice that got IFA alone (FIG. 4E). The addition ofBl-Eng2 to the calnexin vaccine formulation also increased survivalsignificantly vs. control mice vaccinated with calnexin alone (FIG. 4E).This is remarkable since the number of Ag-specific T cell precursorsbefore vaccination was far lower in the absence than in the presence oftransferred of naïve 1807 cells, indicating that Bl-Eng2 is a powerfuladjuvant that drives the development of protective endogenouscalnexin-specific CD4⁺ T cells (FIG. 4F).

Bl-Eng-2 Augments In Vivo Killing of Fungi by Neutrophils (PMN) andAlveolar Macrophages—

To investigate the downstream myeloid effector mechanisms of Bl-Eng-2adjuvancy we used red fluorescent B. dermatitidis yeast to reportphagocytic uptake and fungal viability during cellular interactions withthe murine leukocytes. The concept of using fluorescence to monitormicrobial fate and investigate functional outcomes of individualmicrobial cell-host cell encounters has been introduced recently andprovides a powerful strategy to measure effector mechanisms in vivo. Atday 4 post-infection, mice vaccinated with calnexin+Bl-Eng-2 andcalnexin+Alum+Bl-Eng-2 showed increased activation and killing byneutrophils and alveolar macrophages vs. calnexin and calnexin+Alumcontrols (FIGS. 5A-5C and FIG. 11). The increase in in vivo fungalkilling by neutrophils and macrophages correlated with reduced numbersof DsRed⁺ yeast in the lung (FIGS. 5D and 11D) and CFU by plating (FIGS.5E and 11C). Bl-Eng-2 mediated effects were observed in the presence ofadoptively transferred 1807 T cells (FIG. 5) and by endogenous CD4⁺ Tcells without adoptive transfer (FIGS. 11C-11D). Thus, the addition ofBl-Eng-2 augments the activation and killing by myeloid effector cellssuch as the neutrophils and alveolar macrophages in the lung.

Discussion

We describe a novel ligand for Dectin-2: Bl-Eng2. Discovery of a potentCLR ligand may address a limitation of current vaccines: the lack ofadjuvants that elicit protective cell-mediated immunity. The approach wetook to identify Bl-Eng2 was based on prior work from our group andother laboratories. Dectin-2 recognizes and mediates host defenseagainst several fungi including C. albicans, C. glabrata, A. fumigatus,Malassezia spp., Coccidiodes posadasii, Histoplasma capsulatum and B.dermatitidis. Additionally, Dectin-2^(−/−) mice vaccinated withattenuated B. dermatitidis yeast fail to prime Ag-specific Th1 and Th17cells or acquire vaccine resistance to pulmonary infection. Thus,Dectin-2 regulates innate recognition of the fungal vaccine, and thedevelopment of a protective cellular immune response. Hence, we soughtto identify the Dectin-2 ligand from the vaccine strain. We hypothesizedthat the ligand would prime APC to produce cytokines (e.g. IL-6) thatare known to foster the development of Th17 cells that protect againstlethal fungal challenge.

By using Dectin-2 reporter cells as a probe, we enriched and identifiedBl-Eng2 by ConA binding, gel filtration and Mass spectrometry. Theidentification of Bl-Eng2 also led us to unveil the unappreciated roleof Asp-Eng2 in binding Dectin-2. Both Eng2 proteins are bona fideDectin-2 ligands since they trigger NFAT signaling in Dectin-2 reportercells. Bl-Eng2 features a 45.2% overall and 60.1% GH16 domain sequencesimilarity to Eng2 from A. fumigatus (Asp-Eng2) and contains aSer/Thr-rich C-terminus that both proteins have in common. Bl-Eng2 andAsp-Eng2 respectively harbor 68 and 74 potential O-linked glycosylationsites within their respective 134-aa and 234-aa long Ser/Thr-richC-terminus, but display no consensus sites for N-linked glycosylation(Asn-X-Ser/Thr). In addition to the Eng2 glycoproteins, we now alsoestablish here that MP98 from C. neoformans serves as a ligand forDectin-2.

Dectin-2 has been reported to recognize high mannose structures offungi, such as α-1,2-mannan from C. albicans and furfurman, which is amannoprotein from Malassezia spp. Man-Lam from M. tuberculosis consistsof four components: a mannosyl-phophatidyl-myo-inositol (MPI) anchor, amannose backbone, an arabinan domain, and a α1,2-mannose cap. MP98 fromC. neoformans is a mannoprotein with a Mr of 98 kDa; it contains 12possible N-linked glycosylation sites, and 103 Ser/Thr residues at theC-terminus that serve as potential 0-linked glycosylation sites. Theminimal unit of Bl-Eng2 that confers ligand activity is uncertain. Sinceboth mannosidase and proteinase K digestion of CWE starting materialreduced Dectin-2 ligand activity, both the protein and glycan moietiesof Bl-Eng2 may contribute to its action, perhaps explaining its superiorstimulation of cytokine responses compared to the other ligands.

We found that recombinant Bl-Eng2 elicits potent downstream functions.It induces the production of IL-6 by BMDC in a Dectin-2- andCard9-dependent manner. In addition, Bl-Eng2 induces the production ofIL-6 and IL-1γ by human PBMC, which may have strong implications for thetranslational aspect of our discovery. In comparison to previouslydescribed Dectin-2 ligands, Bl-Eng2 triggers superior cytokineproduction by murine BMDC. Ligand induced IL-6 production was >100 foldhigher for Bl-Eng2 than the other Dectin-2 ligands: Furfurman fromMalassezia spp. and Mannose-capped lipoarabinomannan (Man-Lam) from M.tuberculosis and MP98 from C. neoformans.

Bl-Eng2 induction of T cell priming cytokines by APCs efficientlypromoted the development of calnexin Ag-specific Th17 cells (more sothan Th1 cells), and recall of these cells to the lung upon fungalchallenge of vaccinated mice. The large numbers of pro-inflammatory Tcells sharply reduced lung CFU and increased survival after infection ofBl-Eng2 vaccinated vs. control mice. In comparison, combining commercialAlum with the calnexin subunit vaccine did not show an adjuvant effect.However, Bl-Eng-2 combined with Alum augmented its adjuvancy indicatingthat Bl-Eng-2 has the potential to improve T cell priming by thecommercially available and FDA approved Alum. Thus, in our subunitvaccine model, Bl-Eng2-induced Dectin-2 signaling was associated withcellular immune responses that protected mice against lethal pulmonaryfungal infection. Although not experimentally addressed in thismanuscript, it is conceivable that Bl-Eng-2 can also augment theinduction of CD4⁺ T cell-dependent antibody responses that promote hostprotection against fungi, especially when combined with Alum since thelatter is known to stimulate both T and B cell immune responses. Itremains to be investigated whether antibody will be protective in ourvaccine setting.

We previously reported that mice vaccinated with calnexin and otheradjuvants (glucan particles engaging Dectin-1, Adjuplex, or thecombination) were optimally protected when we adoptive transferred naïve1807 cells to increase the pool of Ag-experienced CD4⁺ T cells. Here,the addition of Bl-Eng2 to the same calnexin vaccine reduced lung CFU bymore than two to three logs vs. control mice even without adoptivetransfer of large numbers of naïve 1807 T cell precursors. These resultsimply that engagement of Dectin-2 by Bl-Eng2 may be better thanengagement of Dectin-1 by glucan particles and other previously usedadjuvants at expanding the pool of endogenous calnexin-specific CD4⁺ Tcell precursors or that Bl-Eng2 induced individual Ag-experienced cellsto produce larger amounts of effector cytokines. Thus, Bl-Eng2 may be apowerful vaccine adjuvant in situations where T cell precursors are lowin number and adoptive transfer of naïve T cell precursors is either notfeasible or too costly.

In contrast to the protective effects of Bl-Eng2 vaccination, Man-Laminduced Dectin-2 responses that caused Th17 cell-mediated autoimmunedisease pathology and EAE. Man-Lam stimulation of Dectin-2 lead to thedevelopment of MOG₃₅₋₅₅ peptide-specific T cells that produced IL-17,IFN-γ and GM-CSF upon ex vivo stimulation. This could simply relate tomodel selection rather than adjuvant efficiency. Thus, it is unclearwhether Man-Lam is capable of inducing protective T cell immunity in aninfectious disease setting. Although C. neoformans MP98 and its glycanmodifications also promoted T cell activation, the T-helper phenotypeand functional role in resistance by primed T cells were notinvestigated.

In conclusion, among the few Dectin-2 ligands reported to date, or newlydiscovered here, Bl-Eng2 is the most potent at stimulating murine andhuman cells to produce cytokines known to foster the development ofprotective Th17 and Th1 cells e.g. IL-6 and IL-1β. The production ofIL-17 and IFN-γ by Th17 and Th1 cells then promotes the activation andkilling of fungi by myeloid effector cells such as neutrophils andalveolar macrophages. Since Bl-Eng2 also greatly augments protectiveimmunity mediated by a subunit vaccine, Bl-Eng2 could potentially beharnessed as an adjuvant for vaccination against infectious disease thatrequires cellular immunity for host defense. The structural basisunderpinning Bl-Eng2 potency as an adjuvant will be important toinvestigate and understand so that those features can be harnessed forvaccine development in the fight against infectious disease due tointracellular pathogens.

Material and Methods

Fungi—

Strains used were wild-type, virulent B. dermatitidis ATCC strain 26199,DsRed26199 and strain #55, the isogenic, attenuated mutant lacking BAD1.B. dermatitidis was grown as yeast on Middlebrook 7H10 agar with oleicacid-albumin complex (Sigma) at 39° C.

Mouse Strains—

Inbred wild type C57BL/6 and congenic B6. PL-Thy1^(a)/Cy (stock #00406)mice carrying the Thy 1.1 allele were obtained from JacksonLaboratories, Bar Harbor, Me. Blastomyces-specific TCR Tg 1807 mice weregenerated in our lab and were backcrossed to congenic Thy1.1⁺ mice asdescribed elsewhere. Dectin-2^(−/−) mice were bred at our facility. Micewere 7-8 weeks old at the time of these experiments. Mice were housedand cared for as per guidelines of the University of Wisconsin AnimalCare Committee who approved all aspects of this work.

Preparation of CWE—

Blastomyces dermatitidis yeast were harvested from 7H10 agar, washedwith H₂O, and sonicated for 3 min on ice. After centrifuging, thesoluble extract was collected, passed through a 0.45-μm pore-size filterand used as CWE. The protein level was measured with the Pierce BCAassay (Thermo Fisher Scientific).

Enrichment of Mannosylated Proteins and Mass Spectrometry Analysis—

To enrich the mannosylated proteins, CWE was incubated with ConcanavalinA (ConA) Sepharose resin (Sigma-Aldrich), and the bound fraction waseluted with methyl-α-D-mannopyranoside as described previously. TheConA-enriched proteins were then applied to a size exclusion column ofUltragel AcA 44 resin (Pall) and eluted with PBS. The ConA enrichmentand size exclusion fractions were assessed using SDS-PAGE and silverstaining. Size exclusion fractions that contained Dectin-2 ligandactivity were analyzed by mass spectrometry as previously described atthe Mass Spectrometry Facility, University of Wisconsin-Madison.Briefly, peptides were analyzed by nano-LC-MS/MS using the Agilent 1100nanoflow system (Agilent Technologies) connected to a hybrid linear iontrap-orbitrap mass spectrometer (LTQ-Orbitrap XL, Thermo FisherScientific) equipped with a nanoelectrospray ion source.

Generation and Purification of r-Bl-Eng2—

Bl-Eng2 was cloned and expressed in P. pastoris using standardrecombinant techniques. Total RNA was extracted from B. dermatitidisyeast and transcribed to cDNA as previously described. Using the cDNA asa template, the Bl-ENG2 coding sequence was amplified using KOD HotStart DNA Polymerase (Toyobo) with primers5′-GGCTCGAGAAAAGAGAGGCTGAAGCTAGGGCTACCAAGCTCGCGTT (SEQ ID NO:9) and5′-GTTTCTAGACCGTACTTGTCATTTGTGGGGTATCCCG (SEQ IN NO:10), and insertedin-frame into the XhoI/XbaI sites of the pPICZαA vector (Invitrogen).The resulting expression vector was then linearized with PmeI andtransformed into Pichia pastoris strain X-33 (Invitrogen) byelectroporation. Yeast colonies were screened for Bl-Eng2 proteinexpression by Western blot analysis using an anti-His antibody (CellSignaling Technology). Bl-Eng2 protein secreted from methanol-inducedyeast cells was purified using Ni-NTA agarose (Qiagen) according to themanufacturer's protocol, and dialyzed against PBS. Purity of recombinantBl-Eng2 was assessed by SDS-PAGE and silver staining. Without beingbound to any particular theory, it is believed that the alpha-factorsignal peptide is excised from the recombinant Bl-Eng2 upon secretion ofthe protein from the yeast. The predicted sequence of the Bl-Eng2recombinant protein after excision of the alpha-factor signal peptide isincluded below.

Predicted recombinant Bl-Eng2 protein: alpha-factor signal peptideexcised during secretion (SEQ ID NO:11).

RATKLALLAALAKLSTGAYVLQDDYQPSNFFDDFAFFDGPDPSNAYVTYVDKSKALRDGLASNNNDFVYLGVDHQNVARGRGRESVRLETKKSYKHGLIVADISHMPGNICGTWPAFWATGATWPDDGEFDIIEGVNKQNKNVVALHTTAGCKVEDNNKYSGILVTKDCDVYSPNQPSNQGCLFRAPSATSYGTAFNSIGGGVYATEWTSDSISVWFFPRYQIPSNINDENPDPSTWPRPIAHFTGCEFDKFFQEQRIIFNTAFCGDWAKATWNENGCAAGGRTCEDYVKNNPWAFSEAFWSINYMKVFQNKQGDTSTSTTTSSTSSTSSSSTEAPTTTMTTSSTYEPSVSSSTAPEPSQSASTPSEYPQPSTAEPTASSSSYPKSSFASTDSPVPTDYPVPSSDEPTVPSATYSESSPVPTDYPVPSSDEPTVPSATYSESLPSASAPSEYPTGTASVDPTDVSSCTPPPTQSCITYTTKTTIAIVVTAPESYKEAIQTESAEDETEPAAYPTEPAGYPTNDKYGLEQKLISEEDLNSAVDHHHHHH

Carbohydrate Analysis—

Bl-Eng2 protein glycosylation was assessed using the Pierce GlycoproteinStaining Kit (Thermo Fisher Scientific). Monosaccharide composition wasdetermined by gas chromatography as described elsewhere.

CLR Reporter Assay—

B3Z/BWZ reporter cells expressing Dectin-2, Mincle, MCL and Dectin-1have been described previously. For B3Z/BWZ cell stimulation, 10⁵B3Z/BWZ cells per well in a 96-well plate were incubated for 18 h withheat-killed fungal cells or plate-coated ligands. β-galactosidase (lacZ)activity was measured in total cell lysates using CPRG (Roche) as asubstrate. OD 560 was measured using OD 620 as a reference.

Stimulation of Mouse BMDCs or Human PBMCs and CytokineDetection—Generation of bone marrow-derived dendritic cells (BMDCs) hasbeen described previously. Peripheral blood mononuclear cells (PBMCs)were isolated from heparinized whole blood collected over Ficoll-PaquePlus (GE). 1-2×10⁵ BMDCs or 5×10⁵ PBMCs per well in a 96-well plate wereincubated with plate-bound Bl-Eng2. After 24 h, supernatants werecollected and cytokine levels were measured by ELISA (R&D Systems orBiolegend) according to the manufacturer's specifications.

Vaccination with Calnexin and Bl-Eng2 and Enumeration of RareEpitope-Specific T Cells—

Prior to vaccination, mice received adoptively transferred naïve 1807 Tcells or not. Mice were vaccinated twice subcutaneously with 10 μgrecombinant calnexin and 10 μg Bl-Eng2 formulated in incomplete Freund'sadjuvant (IFA), two weeks apart. Two weeks after the boost, mice werechallenged with 2×10⁴ 26199 yeast and analyzed for lung T cell responses(at day 4 post-infection) and lung CFU (at day 4 or two weekspost-infection). 1807 T cell responses were detected with the congenicThy1.1 marker and endogenous, calnexin-specific T cells by tetramer. Tcells were detected using the following antibodies: tetramer-PE,CD4-BUV395, CD8-PeCy7, CD3-BV421, CD90.2-BV785, CD44-BV650, Live-deadNear IR, IFN-γ-A488 and IL-17-A647.

Intracellular Cytokine Stain—

Lung cells were harvested at day 4 post-infection. Cells (0.5×10⁶cells/ml) were stimulated for 5 hours with anti-CD3 (clone 145-2C11; 0.1μg/ml) and anti-CD28 (clone 37.51; 1 μg/ml) in the presence ofGolgi-Stop (BD Biosciences). Stimulation with fungal ligands yieldedcomparable cytokine production by transgenic T-cells compared toCD3/CD28 stimulation. After cells were washed and stained for surfaceCD4 and CD8 using anti-CD4 BV395, anti-CD8 PeCy7, and anti-CD44-FITCmAbs (Pharmingen), they were fixed and permeabilized in Cytofix/Cytopermat 4° C. overnight. Permeabilized cells were stained with anti-IL-17A PEand anti-IFN-γ Alexa 700 (clone XMG1.2) conjugated mAbs (Pharmingen) inFACS buffer for 30 min at 4° C., washed, and analyzed by FACS. Cellswere gated on CD4 and cytokine expression in each gate analyzed. Thenumber of cytokine positive CD4⁺ T cells per lung was calculated bymultiplying the percent of cytokine-producing cells by the number ofCD4⁺ T cells in the lung.

The Generation of Bone Marrow Dendritic Cells—

Bone marrow-derived dendritic cells (BMDCs) were obtained from thefemurs and tibias of individual mice. Each bone was flushed with 10 mlof 1% FBS in RPMI through a 22G needle. Red blood cells were lysedfollowed by wash and re-suspension of cells in 10% FBS in RPMI medium.In a petri dish, 2×10⁶ bone marrow cells were plated in 10 ml of RPMIcontaining 10% FBS plus penicillin-streptomycin (P/S) (HyClone),2-mercaptoethanol and 20 ng/ml of rGM-CSF. The culture media wererefreshed every three days and BMDCs were harvested after 10 days for invitro co-culture assays.

Ex vivo stimulation of primed T cells for cytokine proteinmeasurement—Ex vivo cell culture supernatants were generated using thebrachial and inguinal draining lymph nodes harvested from mice 28 dayspost-vaccination and at day 4 post-infection, washed and resuspended incomplete RPMI containing 10 μg/ml recombinant calnexin, and plated in96-well plates at a concentration of 5×10⁵ cells/well. Supernatants werecollected from ex vivo co-cultures after three days of incubation at 37°C. and 5% CO₂. IFN-γ and IL-17 (R&D System) were measured by ELISAaccording to manufacturer specifications (detection limits, 0.05 ng/mland 0.02 ng/ml, respectively).

Tracking Association of Yeast with Neutrophils and Alveolar MacrophagesIn Vivo—

Mice were euthanized three days after challenge i.t. with 10⁵ DsRedyeast and hearts were perfused with PBS to remove blood from the lungsto improve staining. Lungs were dissociated, digested and stained asdescribed previously. In summary, lungs were dissociated and digested inbuffer containing collagenase D and DNase I. After erythrocyte lysis,cells were stained for myeloid cell markers and then fixed inCytofix/Cytoperm (BD Biosciences, San Jose, Calif.). Cells were stainedfor 30 minutes at room temperature with 1 μg/ml Uvitex-2B (Polysciences,Warrington, Pa.) diluted in BD perm/wash buffer and then subsequentlywashed with BD perm/wash buffer and fixed with 2% paraformaldehyde.

Statistics—

Differences in the number of cells and lung CFU were analyzed usingWilcoxon rank and Mann Whitney test for non-parametric data or a T-testif data were normally distributed. A Bonferroni adjustment was used tocorrect for multiple tests. A value of P<0.05 is considered significant.

Example 2

The embodiment described herein demonstrates the use of Bl-Eng2 as avaccine adjuvant in bacterial and viral vaccines. As demonstrated inFIG. 12 and FIG. 13, Bl-Eng2 functions as an adjuvant in bacterial andviral vaccine formulations and increased the number of activated (CD44+)CD4+ and CD8+ producing T cells. Mice were vaccinated with Ag85B fromMycobacterium tuberculosis and Nucleoprotein (NP) from Influenza A inthe presence and absence of Bl-Eng-2 and compared the number ofcorresponding cytokine producing CD4⁺ and CD8⁺ T cells, respectively. Inthe TB vaccine model, Ag-specific IL-17 and IFN-γ producing CD4⁺ T cellsand in the Influenza model, IFN-γ producing cytotoxic CD8⁺ T cells (CTL)are thought to be most protective against bacterial and viral infection,respectively. The addition of Bl-Eng-2 to Ags 85B or NP augmented thenumber of cytokine producing Ag-specific CD4⁺ and CD8⁺ T cells in bothmodels. Thus, Bl-Eng-2 augments immunity also in response to non-fungal(bacterial and viral) Ags and does not only increase CD4⁺ but also CD8⁺T cell immune responses.

Example 3

The embodiment described herein demonstrates the use of Bl-Eng2 as anovel antigen for use in vaccine compositions. As demonstrated in FIGS.14A-14B and FIGS. 15A-15B, Bl-Eng2 functions as an antigen when used invaccine compositions to raise antifungal T cells in the subject mice.Mice subcutaneously vaccinated with Bl-Eng2 formulated in IFA hadsignificantly reduced lung CFU at 4 (15-fold) and 11 post-infection(>5,000 fold) compared to control mice (FIG. 14). Splenocytes fromBl-Eng2 vaccinated mice produced >10 ng/ml INF-γ when stimulated invitro with full length Bl-Eng2 protein or peptide 1 which is comprisedof the following amino acid sequence: AFFDGPDPSNAYV (SEQ ID NO:4).Therefore vaccination with this peptide alone will engender a similarlevel of protection as full length Bl-Eng2 protein.

This peptide could also be used to expand autologous, endogenousBl-Eng2-specific T cells of patients that will undergo transplantation,chemotherapy or other immunocompromising treatments to boost theirimmunity against fungal infection (e.g. cellular immunotherapy followingstem cell transplantation). The lethality of invasive pulmonaryinfection with Aspergillus fumigatus is 50-90% in that patientpopulation.

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We claim:
 1. A vaccine suitable to immunize a patient comprising anadjuvant, wherein the adjuvant is a Dectin-2 ligand.
 2. The vaccine ofclaim 1, wherein the Dectin-2 ligand is a glycoprotein.
 3. The vaccineof claim 1, wherein the Dectin-2 ligand is Bl-Eng2.
 4. The vaccine ofclaim 3, wherein Bl-Eng2 comprises SEQ ID NO:1.
 5. The vaccine of claim3, wherein Bl-Eng2 comprises O-linked glycosylations.
 6. The vaccine ofclaim 1, wherein the vaccine immunizes a patient against a fungalinfection.
 7. The vaccine of claim 1, wherein the vaccine comprisesglucan particles.
 8. The vaccine of claim 1, wherein the vaccineimmunizes a patient against a bacterial infection.
 9. The vaccine ofclaim 1, wherein the vaccine immunizes a patient against a viralinfection.
 10. A method of preparing a vaccine comprising the steps of,(a) preparing a pharmaceutically acceptable vaccine stabilizer; and (b)introducing to the vaccine stabilizer a suitable antigen and anadjuvant, wherein the adjuvant is a Dectin-2 ligand.
 11. A method ofprotecting a patient from an infection comprising the steps of: (a)obtaining the vaccine of claim 1, wherein the vaccine comprises anadjuvant and a suitable antigen, wherein the adjuvant is a Dectin-2ligand; and (b) providing a therapeutically effective amount of thevaccine to a subject, wherein the subject is protected from theinfection.
 12. The method of claim 11 wherein the infection is a fungalinfection and the patient is protected from a fungi infection.
 13. Themethod of claim 12, wherein the antigen is a fragment of calnexin andthe fungi is selected form the group consisting of Histoplasma,Coccidiodes, Paracoccidioides, Penicillium, Blastomyces, Sporothrix,Aspergillus, Pneumocystis, Magnaportha, Exophiala, Neuroaspora,Cryptococcus, Schizophyllum, and Candida.
 14. A vaccine compositioncomprising Bl-Eng2 and a pharmaceutically acceptable carrier.
 15. Thevaccine of claim 14, wherein Bl-Eng2 comprises SEQ ID NO:1.
 16. Thevaccine of claim 14, wherein Bl-Eng2 comprises O-linked glycosylations.17. The vaccine of claim 14, wherein the vaccine is suitable to immunizea subject against a fungal infection.
 18. The vaccine of claim 14,wherein the vaccine additionally comprises an adjuvant.
 19. The vaccineof claim 18, wherein the vaccine comprises incomplete Freunds adjuvant.20. The vaccine of claim 14, wherein the vaccine comprises a fragment ofBl-Eng2.
 21. A method of protecting a patient from an infectioncomprising the steps of: (a) obtaining the vaccine of claim 14, whereinthe vaccine comprises Bl-Eng2; and (b) providing a therapeuticallyeffective amount of the vaccine to a subject, wherein the subject isprotected from the infection.
 22. The method of claim 21, wherein theinfection is a fungal infection.
 23. The method of claim 21, whereinBl-Eng2 comprises SEQ ID NO:1.
 24. The vaccine of claim 21, whereinBl-Eng2 comprises O-linked glycosylations.